The DMI1, DMI2, and DMI3 genes of Medicago truncatula, which are required for both nodulation and mycorrhization, control early steps of Nod factor signal transduction. Here, we have used diverse approaches to pave the way for the map-based cloning of these genes. Molecular amplification fragment length polymorphism markers linked to the three genes were identified by bulked segregant analysis. Integration of these markers into the general genetic map of M. truncatula revealed that DMI1, DMI2, and DMI3 are located on linkage groups 2, 5, and 8, respectively. Cytogenetic studies using fluorescent in situ hybridization (FISH) on mitotic and pachytene chromosomes confirmed the location of DMI1, DMI2, and DMI3 on chromosomes 2, 5, and 8. FISH-pachytene studies revealed that the three genes are in euchromatic regions of the genome, with a ratio of genetic to cytogenetic distances between 0.8 and 1.6 cM per microm in the DMI1, DMI2, and DMI3 regions. Through grafting experiments, we showed that the genetic control of the dmi1, dmi2, and dmi3 nodulation phenotypes is determined at the root level. This means that mutants can be transformed by Agrobacterium rhizogenes to accelerate the complementation step of map-based cloning projects for DMI1, DMI2, and DMI3.

            The Medicago Genome Initiative (MGI) is a database of EST sequences of the model legume MEDICAGO: truncatula. The database is available to the public and has resulted from a collaborative research effort between the Samuel Roberts Noble Foundation and the National Center for Genome Resources to investigate the genome of M.truncatula. MGI is part of the greater integrated MEDICAGO: functional genomics program at the Noble Foundation (http://www.noble.org ), which is taking a global approach in studying the genetic and biochemical events associated with the growth, development and environmental interactions of this model legume. Our approach will include: large-scale EST sequencing, gene expression profiling, the generation of M.truncatula activation-tagged and promoter trap insertion mutants, high-throughput metabolic profiling, and proteome studies. These multidisciplinary information pools will be interfaced with one another to provide scientists with an integrated, holistic set of tools to address fundamental questions pertaining to legume biology. The public interface to the MGI database can be accessed at http://www.ncgr.org/research/mgi.

            The genome sequence of Arabidopsis is complete and the genomes of plants representing legumes (Medicago truncatula) and grasses (rice) will soon follow. The rate at which new genes have been discovered has far outstripped the pace at which their function is determined. The greatest hurdle that plant biologists face in assigning gene function and in crop improvement is the lack of efficient and robust technologies to generate gene replacements or targeted gene knockouts. Many of the factors underlying these events remain to be elucidated. This review addresses the current status of plant gene targeting and what is known about the associated plant DNA repair mechanisms.

            Apyrases have been suggested to play important roles in plant nutrition, photomorphogenesis, and nodulation. To help trace the evolution of these genes in the legumes-and possibly, the acquisition of new functions for nodulation-apyrase-containing BACs were sequenced from three legume genomes. Genomic sequences from Medicago truncatula, Glycine max and Lotus japonicus were compared to one another and to corresponding regions in Arabidopsis thaliana. A phylogenetic analysis of apyrase homologs from these regions and sequences from other legume species, as well as other plant families, identified a potentially legume-specific clade that contains a well-characterized soybean ( G. soja) apyrase, Gs52, as well as homologs from Dolichos, Lotus, Medicago and Pisum. Sister clades contain homologs from members of Brassicaceae, Solanaceae, Poaceae and Fabaceae. Comparisons of rates of change at synonymous and nonsynonymous sites in the Gs52 and sister clades show rapid evolution in the potentially legume-specific Gs52 clade. The genomic organization of the apyrase-containing BACs shows evidence of gene duplication, genomic rearrangement, and gene conversion among Gs52 homologs. Taken together, these results suggest a scenario of local apyrase gene duplication in an ancestor of the legumes, followed by functional diversification and increased rates of change in the new genes, and further duplications in the Galegae (which include the genera Medicago and Pisum). The study also provides a detailed comparison of genomic regions between two model genomes which are now being sequenced ( M. truncatulaand L. japonicus), and a genome from an economically important legume species ( G. max).

            We have characterized from the legume plant Medicago a new family of miniature inverted-repeat transposable elements (MITE), called the Bigfoot transposable elements. Two of these insertion elements are present only in a single allele of two different M. sativa genes. Using a PCR strategy we have isolated 19 other Bigfoot elements from the M. sativa and M. truncatula genomes. They differ from the previously characterized MITEs by their sequence, a target site of 9 bp and a partially clustered genomic distribution. In addition, we show that they exhibit a significantly stable secondary structure. These elements may represent up to 0.1% of the genome of the outcrossing Medicago sativa but are present at a reduced copy number in the genome of the autogamous M. truncatula plant, revealing major differences in the genome organization of these two plants.

            Rhizobium meliloti can interact symbiotically with Medicago plants, thereby inducing root nodules. However, certain Medicago plants can form nodules spontaneously, in the absence of rhizobia. A differential screening was performed using spontaneous nodule versus root cDNAs from Medicago sativa ssp. varia. Transcripts of a differentially expressed clone, Msenod40, were detected in all differentiating cells of nodule primordia and spontaneous nodules, but were absent in fully differentiated cells. Msenod40 showed homology to a soybean early nodulin gene, Gmenod40, although no significant open reading frame (ORF) or coding capacity was found in the Medicago sequence. Furthermore, in the sequences of cDNAs and a genomic clone (Mtenod40) isolated from Medicago truncatula, a species containing a unique copy of this gene, no ORFs were found either. In vitro translation of purified Mtenod40 transcripts did not reveal any protein product. Evaluation of the RNA secondary structure indicated that both msenod40 and Gmenod40 transcripts showed a high degree of stability, a property shared with known non-coding RNAs. The Mtenod40 RNA was localized in the cytoplasm of cells in the nodule primordium. Infection with Agrobacterium tumefaciens strains bearing antisense constructs of Mtenod40 arrested callus growth of Medicago explants, while overexpressing Mtenod40 embryos developed into teratomas. These data suggest that the enod40 genes might have a role in plant development, acting as 'riboregulators', a novel class of untranslated RNAs associated with growth control and differentiation.

            Medicago truncatula Gaertn. is an annual self-pollinating species characterized by a diploid complement 2n = 16 and low DNA content. It responds very well to transformation methods so it is used as a model species for Leguminosae. In contrast with the advanced studies in molecular biology, cytogenetic research has remained limited even though it is an extremely valuable approach to the analysis of the genome structure. In the present study we examined the chromosomal distribution of rDNA sequences in five natural populations of M. truncatula, explored the genomic diversity of this species and found markers for chromosome identification. FISH experiments revealed three distribution patterns of rDNA sequences, distinguished by one, two and three loci of 5S genes; 18S-5.8S-25S genes were always localized at a single locus. The results add information to the genome structure of M. truncatula, revealing a pattern of distribution of rDNA genes unobserved previously, which consists of 5S genes clustered at a single locus. The physical mapping of rDNA sequences is a first contribution towards the construction of a detailed molecular karyotype of M. truncatula.

            The Medicago truncatula expressed sequence tag (EST) database (Gene Index) contains over 140,000 sequences from 30 cDNA libraries. This resource offers the possibility of identifying previously uncharacterized genes and assessing the frequency and tissue specificity of their expression in silico. Because M. truncatula forms symbiotic root nodules, unlike Arabidopsis, this is a particularly important approach in investigating genes specific to nodule development and function in legumes. Our analyses have revealed 340 putative gene products, or tentative consensus sequences (TCs), expressed solely in root nodules. These TCs were represented by two to 379 ESTs. Of these TCs, 3% appear to encode novel proteins, 57% encode proteins with a weak similarity to the GenBank accessions, and 40% encode proteins with strong similarity to the known proteins. Nodule-specific TCs were grouped into nine categories based on the predicted function of their protein products. Besides previously characterized nodulins, other examples of highly abundant nodule-specific transcripts include plantacyanin, agglutinin, embryo-specific protein, and purine permease. Six nodule-specific TCs encode calmodulin-like proteins that possess a unique cleavable transit sequence potentially targeting the protein into the peribacteroid space. Surprisingly, 114 nodule-specific TCs encode small Cys cluster proteins with a cleavable transit peptide. To determine the validity of the in silico analysis, expression of 91 putative nodule-specific TCs was analyzed by macroarray and RNA-blot hybridizations. Nodule-enhanced expression was confirmed experimentally for the TCs composed of five or more ESTs, whereas the results for those TCs containing fewer ESTs were variable.

            Glutathione (GSH) and homo-GSH (hGSH) are the major low-molecular weight thiols synthesized in Medicago truncatula. Two M. truncatula cDNAs (gshs1 and gshs2) corresponding to a putative GSH synthetase (GSHS) and a putative hGSH synthetase (hGSHS) were characterized. Heterologous expression of gshs1 and gshs2 cDNAs in an Escherichia coli strain deficient in GSHS activity showed that GSHS1 and GSHS2 are a GSHS and an hGSHS, respectively. Leucine-534 and proline-535 present in hGSHS were substituted by alanines that are conserved in plant GSHS. These substitutions resulted in a strongly stimulated GSH accumulation in the transformed E. coli strain showing that these residues play a crucial role in the differential recognition of beta-alanine and glycine by hGSHS. Phylogenetic analysis of GSHS2 and GSHS1 with other eukaryotic GSHS sequences indicated that gshs2 and gshs1 are the result of a gene duplication that occurred after the divergence between Fabales, Solanales, and Brassicales. Analysis of the structure of gshs1 and gshs2 genes shows they are both present in a cluster and in the same orientation in the M. truncatula genome, suggesting that the duplication of gshs1 and gshs2 occurred via a tandem duplication.

            We have screened a large tomato EST database against the Arabidopsis genomic sequence and report here the identification of a set of 1025 genes (referred to as a conserved ortholog set, or COS markers) that are single or low copy in both genomes (as determined by computational screens and DNA gel blot hybridization) and that have remained relatively stable in sequence since the early radiation of dicotyledonous plants. These genes were annotated, and a large portion could be assigned to putative functional categories associated with basic metabolic processes, such as energy-generating processes and the biosynthesis and degradation of cellular building blocks. We further demonstrate, through computational screens (e.g., against a Medicago truncatula database) and direct hybridization on genomic DNA of diverse plant species, that these COS markers also are conserved in the genomes of other plant families. Finally, we show that this gene set can be used for comparative mapping studies between highly divergent genomes such as those of tomato and Arabidopsis. This set of COS markers, identified computationally and experimentally, may further studies on comparative genomes and phylogenetics and elucidate the nature of genes conserved throughout plant evolution.

            Two leghaemoglobin genes from the diploid, autogamous Medicago truncatula (Mtlb1 and Mtlb2) have been cloned and their nucleotide sequences determined. The deduced amino acid sequences encoded by these two genes differ significantly (18%), confirming that they belong to different sub-groups of Medicago leghaemoglobin genes. RNAse protection experiments have been used to show that both genes are transcriptionally active, and are expressed specifically in the nitrogen-fixing root nodule of M. truncatula. Whilst Mtlb1 mRNA is present at approximatively 3-fold higher steady-state levels than Mtlb2 mRNA, the transcription of both genes is triggered concomitantly during nodule development (5 days after inoculation with Rhizobium meliloti), and the ratio of the steady-state levels of the two mRNA species remains constant throughout nodule maturation. When the growth medium of nodulated M. truncatula is supplemented with 5 mM KNO3 over a period of 2-3 days there is a progressive drop in specific nitrogen fixation activity to only 20-25% of the original level. This is accompanied with a parallel and synchronous reduction in the quantities of mRNA corresponding to both Mtlb1 and Mtlb2. By contrast, the expression of the nodule parenchyma-specific gene ENOD2 is not significantly modified following nitrate treatment, clearly demonstrating differences in tissue-specific gene regulation in response to combined nitrogen.

            A synteny based positional cloning approach was started to clone the pea SYM2 gene by using locally conserved genome structure with the model plant Medicago truncatula. We reported that a pea marker tightly linked to SYM2 was used to screen a M. truncatula BAC library, and two contigs named C1/C2 and C3 were constructed that are both located on the long arm of M. truncatula chromosome 5 and separated by 9 cM. C1/C2 is highly microsyntenic to the pea SYM2 genomic region and corresponds to the M. truncatula SYM2-orthologous region, which is delimitated to 350 kbp. In this manuscript we analyze the distribution in the three contigs of 22 sequences and their homologues, including eight C1/C2 and two pea RFLP markers linked to SYM2. Among the analyzed sequences are several different (receptor) kinase-like gene sequences and two classes of LRR-containing resistance protein-like sequences. From all the studied sequences only four detected homologous sequences in C3, and their distribution is comparable in C1/C2 and C3, suggesting that a 70-kbp and a 120-kbp segments of these two contigs, respectively, arose through a duplication. The implications of these findings for the cloning of SYM2 are discussed.

            The crop legume pea (Pisum sativum) is genetically well characterized. However, due to its large genome it is not amenable to efficient positional cloning strategies. The purpose of this study was to determine if the model legume Medicago truncatula, which is a close relative of pea, could be used as a reference genome to facilitate the cloning of genes identified based on phenotypic and genetic criteria in pea. To this end, we studied the level of microsynteny between the SYM2 region of pea and the orthologous region in M. truncatula. Initially, a marker tightly linked to SYM2 was isolated by performing differential RNA display on near-isogenic pea lines. This marker served as the starting point for construction of a BAC physical map in M. truncatula. A fine-structure genetic map, based on eight markers from the M. truncatula physical map, indicates that the two genomes in this region share a conserved gene content. Importantly, this fine structure genetic map clearly delimits the SYM2-containing region in pea and the SYM2-orthologous region in M. truncatula, and should provide the basis for cloning SYM2. The utility of the physical and genetic tools in M. truncatula to dissect the SYM2 region of pea should have important implications for other gene cloning experiments in pea, in particular where the two genomes are highly syntenic within the region of interest.

            We have isolated and sequenced a sucrose synthase (SucS) cDNA from the model legume Medicago truncatula. This cDNA (MtSucS1) contains an ORF of 2418 bp, coding for a protein of 805 amino acids with a molecular mass of 92.29 kDa. The deduced amino acid sequence shows significant homology to other plant sucrose synthases, in particular to the nodule-enhanced sucrose synthases from pea and broad bean. Northern analysis revealed that the corresponding gene shows a ten-fold higher expression level in root nodules than in uninfected root, stem and leaf tissues. SucS protein was detected in root nodules from a variety of legumes, including M. truncatula. Whereas only one SucS isoform was detectable in root nodules, an additional sucrose synthase of slightly larger molecular weight was present in uninfected root, stem and flower tissues of M. truncatula. From our expression and sequence data we infer that the MtSucS1 gene encodes a nodule-enhanced sucrose synthase in M. truncatula. Southern hybridization data indicate that MtSucS1 is a single-copy gene. An analysis of a genomic MtSucS1 sequence revealed that the gene consists of 14 exons with the start codon being located on exon II. As is common for SucS genes, the MtSucS1 gene contains a large intron of 747 bp in the 5' untranslated region. The transcriptional start of MtSucS1 was mapped and putative regulatory elements in the MtSucS1 promoter were identified.

            The genome of the model legume Medicago truncatula Gaertn. was screened for the presence of genes encoding tonoplast intrinsic proteins, and a gene family was identified. The cDNA fragments of two members of the multigene family were cloned from roots inoculated with an arbuscular mycorrhizal fungus. Transcript accumulation in roots could be detected for both cDNA fragments, but only one gene was induced in the symbiosis when compared to non-mycorrhizal control roots. A full-length cDNA clone was obtained from the arbuscular-mycorrhiza-regulated gene, and injection of in-vitro-transcribed RNA into Xenopus oocytes revealed that the encoded protein MtAQP1 specifically facilitates water transport. The possible role of MtAQP1 in buffering osmotic fluctations in the highly compartmented vacuole of arbuscule cells is discussed.

            In order to identify the genes and gene functions that underlie key aspects of legume biology, researchers have selected the cool season legume Medicago truncatula (Mt) as a model system for legume research. A set of >170 000 Mt ESTs has been assembled based on in-depth sampling from various developmental stages and pathogen-challenged tissues. MtDB is a relational database that integrates Mt transcriptome data and provides a wide range of user-defined data mining options. The database is interrogated through a series of interfaces with 58 options grouped into two filters. In addition, the user can select and compare unigene sets generated by different assemblers: Phrap, Cap3 and Cap4. Sequence identifiers from all public Mt sites (e.g. IDs from GenBank, CCGB, TIGR, NCGR, INRA) are fully cross-referenced to facilitate comparisons between different sites, and hypertext links to the appropriate database records are provided for all queries' results. MtDB's goal is to provide researchers with the means to quickly and independently identify sequences that match specific research interests based on user-defined criteria. The underlying database and query software have been designed for ease of updates and portability to other model organisms. Public access to the database is at http://www.medicago.org/MtDB.

            We are building a framework physical infrastructure across the soybean genome by using SSR (simple sequence repeat) and RFLP (restriction fragment length polymorphism) markers to identify BACs (bacterial artificial chromosomes) from two soybean BAC libraries. The libraries were prepared from two genotypes, each digested with a different restriction enzyme. The BACs identified by each marker were grouped into contigs. We have obtained BAC- end sequence from BACs within each contig. The sequences were analyzed by the University of Minnesota Center for Computational Genomics and Bioinformatics using BLAST algorithms to search nucleotide and protein databases. The SSR-identified BACs had a higher percentage of significant BLAST hits than did the RFLP-identified BACs. This difference was due to a higher percentage of hits to repetitive-type sequences for the SSR-identified BACs that was offset in part, however, by a somewhat larger proportion of RFLP-identified significant hits with similarity to experimentally defined genes and soybean ESTs (expressed sequence tags). These genes represented a wide range of metabolic functions. In these analyses, only repetitive sequences from SSR-identified contigs appeared to be clustered. The BAC-end sequences also allowed us to identify microsynteny between soybean and the model plants Arabidopsis thaliana and Medicago truncatula. This map-based approach to genome sampling provides a means of assaying soybean genome structure and organization.

            Polygalacturonase (PG) is one of the most important enzymes associated with plant cell wall degradation. It has been proposed to participate in the early steps of the Rhizobium-legume interaction. We have identified two classes of cDNA fragments corresponding to two classes of PG genes in the Medicago genome. One of this class, represented by E2 in M. truncatula and Pl1 in M. sativa, seems to be related to previously characterized plant PG genes expressed in pollen. We have isolated the genomic clone containing the entire gene corresponding to the second class (E3). We showed that MsPG3 is a single gene in the Medicago genome coding for PG. By reverse transcription-PCR, MsPG3 expression was detected in roots 1 day after Rhizobium inoculation. The early induction of the MsPG3, as also seen by in situ hybridization experiments, supports its involvement in the early stages of the Rhizobium-legume infection process. In addition, by analyzing the expression of a MsPG3 promoter-gus construct in Vicia hirsuta-transgenic root nodules, we showed that MsPG3 was expressed in all cells of nodule primordia and in the cells of the invasion zone. By Northern blot, MsPG3 transcripts are not detected in various Medicago tissues, indicating that the function of this gene is related closely to symbiosis. Thus, our results strongly suggest the involvement of MsPG3 gene during meristem formation and/or in the infection process, probably by facilitating cell wall rearrangement, penetration of the bacteria through the root hair wall, or infection thread formation and release of bacteria in plant cells. MsPG3 represents a class of PG genes, distinct from the pollen-specific genes, and it is the first pectic encoded enzyme demonstrated to be involved in Rhizobium-legume symbiosis.

            4th Workshop on Medicago truncatula, 7-10 July 2001, Madison WI, USA.

            Primary expression of the Rhizobium meliloti-induced peroxidase gene rip1 occurs prior to nodule morphogenesis, specifically at the site of impending rhizobial infection (D. Cook, D. Dreyer, D. Bonnet, M. Howell, E. Nony, K. VandenBosch [1995] Plant Cell 7: 43-55). We examined the distribution and structure of rip1 transcript throughout nodule development. We determined that expression of rip1 in root tips is correlated with the competence of this zone for symbiotic association, whereas after rhizobial infection rip1 transcript is specifically associated with the zone of nodule development, including nascent nodule primordia. rip1 transcripts are characterized by multiple polyadenylation sites distributed within 200 to 400 bp of the translation stop site, and a single major transcription initiation site in close proximity to the rip1 open reading frame. Thus, rip1 expression is likely to be mediated through effects on a single transcription unit. Immediately 5' of the rip1 transcription unit DNA sequence analysis identified a 377-bp DNA element containing extensive repeat structure that is widely distributed in the Medicago truncatula genome.

            Seventy-three isolates of rhizobia sampled from root nodules of Medicago truncatula were analyzed by restriction fragment length polymorphism (RFLP) of DNA regions amplified by the polymerase chain reaction (PCR) targeting the symbiotic plasmid (nifD-K, nodD1, and nodD2 genes) and the chromosome (16S rDNA plus intergenic spacer). Two genotypic groups were found, regardless of the DNA region targeted. These two groups were given the status of genomic species based on results of DNA/DNA hybridization.

            The glutamine synthetase (GS) gene family of Medicago truncatula Gaertn. contains three genes related to cytosolic GS (MtGSa, MtGSb, and MtGSc), although one of these (MtGSc) appears not to be expressed. Sequence analysis suggests that the genes are more highly conserved interspecifically rather than intraspecifically: MtGSa and MtGSb are more similar to their homologs in Medicago sativa and Pisum sativum than to each other. Studies in which gene-specific probes are used show that both MtGSa and MtGSb are induced during symbiotic root nodule development, although not coordinately. MtGSa is the most highly expressed GS gene in nodules but is also expressed to lower extents in a variety of other organs. MtGSb shows higher levels of expression in roots and the photosynthetic cotyledons of seedlings than in nodules or other organs. In roots, both genes are expressed in the absence of an exogenous nitrogen source. However the addition of nitrate leads to a short-term, 2- to 3-fold increase in the abundance of both mRNAs, and the addition of ammonium leads to a 2-fold increase in MtGSb mRNA. The nitrogen supply, therefore, influences the expression of the two genes in roots, but it is clearly not the major effector of their expression. In the discussion section, the expression of the GS gene family of the model legume M. truncatula is compared to those of other leguminous plants.

            We analyzed 314,254 soybean expressed sequence tags (ESTs), including 29,540 from our laboratory and 284,714 from GenBank. These ESTs were assembled into 56,147 unigenes. About 76.92% of the unigenes were homologous to genes from Arabidopsis thaliana ( Arabidopsis). The putative products of these unigenes were annotated according to their homology with the categorized proteins of Arabidopsis. Genes corresponding to cell growth and/or maintenance, enzymes and cell communication belonged to the slow-evolving class, whereas genes related to transcription regulation, cell, binding and death appeared to be fast-evolving. Soybean unigenes with no match to genes within the Arabidopsis genome were identified as soybean-specific genes. These genes were mainly involved in nodule development and the synthesis of seed storage proteins. In addition, we also identified 61 genes regulated by salicylic acid, 1,322 transcription factor genes and 326 disease resistance-like genes from soybean unigenes. SSR analysis showed that the soybean genome was more complex than the Arabidopsis and the Medicago truncatula genomes. GC content in soybean unigene sequences is similar to that in Arabidopsis and M. truncatula. Furthermore, the combined analysis of the EST database and the BAC-contig sequences revealed that the total gene number in the soybean genome is about 63,501.

            PsCyp15a is a gene that encodes a vacuolar cysteine protease expressed in wilt-induced shoots of Pisum sativum (pea) and in root nodules. To further the understanding of nodular PsCyp15a expression, a region 5' to the coding sequence of the gene was cloned. Varying lengths of 5' untranslated sequence were fused with the uidA coding region and introduced from Agrobacterium rhizogenes into "hairy roots" of Vicia hirsuta. In this transgenic root nodulation assay, a promoter sequence of 900 bp was sufficient to give an expression pattern indistinguishable from that obtained in pea nodules by in situ hybridization. An orthologue of PsCyp15a was cloned from nodule mRNA of Medicago sativa and a corresponding gene identified in M. truncatula was also shown to express strongly in nodules. With molecular mapping techniques, it was demonstrated that these genes map to a syntenic genome location in pea and Medicago spp., but the map positions of the Cyp15a genes cannot be correlated with existing nodulation mutants.

            Isopentenyl diphosphate, the universal precursor of isoprenoids, is synthesized by two separate routes, one in the cytosol and the other in plastids. The initial step of the plastidial pathway is catalysed by 1-deoxy-d-xylulose 5-phosphate synthase (DXS), which was previously thought to be encoded by a single-copy gene. We have identified two distinct classes of DXS-like cDNAs from the model legume Medicago truncatula. The deduced mature MtDXS1 and MtDXS2 proteins, excluding the predicted plastid-targeting peptides, are similar in size (72.7 and 71.2 kDa) yet share only 70% identity in their amino acid sequences, and both encode functional DXS proteins as shown by heterologous expression in Escherichia coli. Available DXS sequences from other plants can easily be assigned to either class 1 or class 2. Partial sequences of multiple DXS genes in a single genome may be found in the databases of several monocot and dicot plants. Blot analyses of RNA from M. truncatula, maize, tomato and tobacco demonstrate preferential expression of DXS1 genes in many developing plant tissues except roots. By contrast, DXS2 transcript levels are low in most tissues but are strongly stimulated in roots upon colonization by mycorrhizal fungi, correlated with accumulation of carotenoids and apocarotenoids. Monoterpene-synthesizing gland cells of leaf trichomes appear to be another site of DXS2 gene activity. The potential importance of DXS1 in many housekeeping functions and a still hypothetical role of DXS2 in the biosynthesis of secondary isoprenoids is discussed.

            A survey of six organ-/tissue-specific proteomes of the model legume barrel medic (Medicago truncatula) was performed. Two-dimensional polyacrylamide gel electrophoresis reference maps of protein extracts from leaves, stems, roots, flowers, seed pods, and cell suspension cultures were obtained. Five hundred fifty-one proteins were excised and 304 proteins identified using peptide mass fingerprinting and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Nanoscale high-performance liquid chromatography coupled with tandem quadrupole time-of-flight mass spectrometry was used to validate marginal matrix-assisted laser desorption ionization time-of-flight mass spectrometry protein identifications. This dataset represents one of the most comprehensive plant proteome projects to date and provides a basis for future proteome comparison of genetic mutants, biotically and abiotically challenged plants, and/or environmentally challenged plants. Technical details concerning peptide mass fingerprinting, database queries, and protein identification success rates in the absence of a sequenced genome are reported and discussed. A summary of the identified proteins and their putative functions are presented. The tissue-specific expression of proteins and the levels of identified proteins are compared with their related transcript abundance as quantified through EST counting. It is estimated that approximately 50% of the proteins appear to be correlated with their corresponding mRNA levels.

            A growing body of research indicates that microsynteny is common among dicot genomes. However, most studies focus on just one or a few genomic regions, so the extent of microsynteny across entire genomes remains poorly characterized. To estimate the level of microsynteny between Medicago truncatula (Mt) and Glycine max (soybean), and also among homoeologous segments of soybean, we used a hybridization strategy involving bacterial artificial chromosome (BAC) contigs. A Mt BAC library consisting of 30,720 clones was screened with a total of 187 soybean BAC subclones and restriction fragment length polymorphism (RFLP) probes. These probes came from 50 soybean contig groups, defined as one or more related BAC contigs anchored by the same low-copy probe. In addition, 92 whole soybean BAC clones were hybridized to filters of HindIII-digested Mt BAC DNA to identify additional cases of cross-hybridization after removal of those soybean BACs found to be repetitive in Mt. Microsynteny was inferred when at least two low-copy probes from a single soybean contig hybridized to the same Mt BAC or when a soybean BAC clone hybridized to three or more low-copy fragments from a single Mt BAC. Of the 50 soybean contig groups examined, 54% showed microsynteny to Mt. The degree of conservation among 37 groups of soybean contigs was also investigated. The results indicated substantial conservation among soybean contigs in the same group, with 86.5% of the groups showing at least some level of microsynteny. One contig group was examined in detail by a combination of physical mapping and comparative sequencing of homoeologous segments. A TBLASTX similarity search was performed between 1,085 soybean sequences on the 50 BAC contig groups and the entire Arabidopsis genome. Based on a criterion of sequence homologues <100 kb apart, each with an expected value of < or =1e-07, seven of the 50 soybean contig groups (14%) exhibited microsynteny with Arabidopsis.

            A growing array of sequence-based tools is helping to reveal the organization, evolution and syntenic relationships of legume genomes. The results indicate that legumes form a coherent taxonomic group with frequent and widespread macro- and microsynteny. This is good news for two model legume systems, Medicago truncatula and Lotus japonicus. Indeed, both models have recently been used to clone and characterize genes for nodulation-related receptors that were originally described in legumes with more complex genomes. Studies of legume genomes have also provided insight into genome size, gene clustering, genome duplications and repetitive elements. To understand legume genomes better, it will be necessary to develop tools for studying under-represented taxa beyond the relatively small group of economically important species that have been examined so far.

            Sequences homologous to the nucleotide binding site (NBS) domain of NBS-leucine-rich repeat (LRR) resistance genes were retrieved from the model legume M. truncatula through several methods. Phylogenetic analysis classified these sequences into TIR (toll and interleukin-1 receptor) and non-TIR NBS subfamilies and further subclassified them into several well-defined clades within each subfamily. Comparison of M. truncatula NBS sequences with those from several closely related legumes, including members of the tribes Trifoleae, Viceae, and Phaseoleae, reveals that most clades contain sequences from multiple legume species. Moreover, sequences from species within the closely related Trifoleae and Viceae tribes (e.g., Medicago and Pisum spp.) tended to be cophyletic and distinct from sequences of Phaseoleae species (e.g., soybean and bean). These results suggest that the origin of major clades within the NBS-LRR family predate radiation of these Papilionoid legumes, while continued diversification of these sequences mirrors speciation within this legume subfamily. Detailed genetic and physical mapping of both TIR and non-TIR NBS sequences in M. truncatula reveals that most NBS sequences are organized into clusters, and few, if any, clusters contain both TIR and non-TIR sequences. Examples were found, however, of physical clusters that contain sequences from distinct phylogenetic clades within the TIR or non-TIR subfamilies. Comparative mapping reveals several blocks of resistance gene loci that are syntenic between M. truncatula and soybean and between M. truncatula and pea.

            Arabidopsis and Medicago truncatula represent sister clades within the dicot subclass Rosidae. We used genetic map-based and bacterial artificial chromosome sequence-based approaches to estimate the level of synteny between the genomes of these model plant species. Mapping of 82 tentative orthologous gene pairs reveals a lack of extended macrosynteny between the two genomes, although marker collinearity is frequently observed over small genetic intervals. Divergence estimates based on non-synonymous nucleotide substitutions suggest that a majority of the genes under analysis have experienced duplication in Arabidopsis subsequent to divergence of the two genomes, potentially confounding synteny analysis. Moreover, in cases of localized synteny, genetically linked loci in M. truncatula often share multiple points of synteny with Arabidopsis; this latter observation is consistent with the large number of segmental duplications that compose the Arabidopsis genome. More detailed analysis, based on complete sequencing and annotation of three M. truncatula bacterial artificial chromosome contigs suggests that the two genomes are related by networks of microsynteny that are often highly degenerate. In some cases, the erosion of microsynteny could be ascribed to the selective gene loss from duplicated loci, whereas in other cases, it is due to the absence of close homologs of M. truncatula genes in Arabidopsis.